THE PHAGOSOMAL ENVIRONMENT PROTECTS VIRULENT MYCOBACTERIUM-AVIUM FROMKILLING AND DESTRUCTION BY CLARITHROMYCIN

Murine bone marrow-derived macrophages (M phi s) infected with virulen t strains of Mycobacterium avium (TMC 724 and 8 human clinical isolate ) or with an avirulent opaque variant that spontaneously dissociates f rom the virulent human clinical isolate were subjected to a prolonged and continuous treatment with clarithromycin added at the MIC. The eff iciency of this antibiotic in terms of inhibition of bacterial growth and bacterial degradation was evaluated during a 21-day treatment peri od. Growth was assessed by determination of CFU of intracellular bacte ria and by a quantitative ultrastructural analysis which allowed us al so to determine the extent of bacterial degradation. A similar treatme nt was applied to the same strains growing in liquid medium. Our data show that in liquid medium, clarithromycin caused a 90% decrease in CF U within 7 days of treatment. When applied to M phi s infected with vi rulent M. avium, clarithromycin immediately arrested bacterial growth but wits unable to fully kill and degrade intracellularly growing viru lent bacteria. After 21 days of treatment, 25% of intracellular bacter ia were still morphologically intact. These bacteria resumed growth op en removal of the antibiotic, with a normal replication rate. These ba cteria had not become more resistant to the drug, since the MIC was un changed as compared to the one determined for the initial stock used t o infect M phi s. Our data therefore suggest that the intraphagosomal environment protects bacteria from degradation, We propose that the in ability of the drug to completely destroy bacteria is the result of a limited accessibility of the drug due to prevention of fusions between the immature phagosomes in which virulent bacteria reside and lysosom es in which clarithromycin accumulates. In accord with our proposal, w e show that the avirulent opaque variant, which does not prevent phago some-lysosome fusions (unpublished data), is finally destroyed by clar ithromycin even within the phagosomal Environment.